Antibodies and plasmids
Mouse monoclonal antibodies to MHCI from hybridoma cell clones w6/32 (immunoglobulin G2a [IgG2a]) were used for immunofluorescence and internalization experiments. Mouse monoclonal antibodies to CD44 (clone BJ18; IgG1), CD55 (clone JS11; IgG1), CD59 (clone p282; IgG1), CD98 (clone MEM-108; IgG1), and CD147 (clone HIM6; IgG1) were purchased from Biolegend (San Diego, CA) and used for immunofluorescence and antibody internalization. Mouse monoclonal antibodies to TfnR (clone 236-15375; IgG1) were purchased from Invitrogen Molecular Probes (Eugene, OR). Goat polyclonal IgG for CD98 (used for Western blotting) and for epsin 1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CD98 (clone MEM-108; IgG1) was used for immunoprecipitations. Rabbit anti-HA was purchased from Covance Research Products (Princeton, NJ). Rabbit anti-FLAG, M2 mouse anti-FLAG, and rabbit anti-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-Lamp1 and rabbit anti–clathrin heavy-chain antibodies were purchased from Abcam (Cambridge, MA). Mouse monoclonal anti-GLUT1 antibody (clone 202915; IgG2B) was purchased from R&D Systems (Minneapolis, MN). Mouse anti-TSG101 antibody was purchased from GeneTex (Irvine, CA). All Alexa-conjugated secondary antibodies were purchased from Invitrogen Molecular Probes (Eugene, OR).
MARCH1-FLAG, MARCH2-FLAG, MARCH3-FLAG, MARCH4-FLAG, and MARCH8-FLAG were as described (Bartee et al., 2004
). HA-ubiquitin was from James Hurley (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD).
HeLa or ARPE-19 cells were grown on glass coverslips (for immunofluorescence), six-well dishes (for flow cytometry), or 35- and 10-cm plates (for whole-cell lysate generation) and transfected using FuGene (Roche, Indianapolis, IN) according to manufacturer's specifications. Transfections with multiple plasmids were performed with 1 μg of each plasmid per transfection. Experiments were carried out 18 h after transfection. TSG101 siRNA (Thermo Scientific, Dharmacon, Chicago, IL) was prepared according to manufacturer's instructions and transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 24 h.
Immunofluorescence and flow cytometry
HeLa or ARPE-19 cells were plated on class coverslips (immunofluorescence) or six-well dishes (flow cytometry) 48 h prior to use. Cells were trypsinized for 10 min and transferred to 2058 Falcon tubes (Becton Dickinson Labware, Franklin Lakes, NJ) (flow cytometry). For antibody internalization experiments, cells were preincubated with primary antibodies (diluted to 10 μg/ml in media) to CD44, CD98, MHCI, TfnR, or Tac for 1 h at 37°C and then transferred to fresh media containing 15–25 mM NH4Cl for 2, 6, 13, or 24 h. Dynasore (Sigma-Aldrich) was prepared in dimethyl sulfoxide and used according to manufacturer's instructions. For internalization experiments, antibodies to CD44, CD98, MHCI, or TfnR were added to cells to allow internalization for indicated times, and surface antibody was removed with 0.5% acetic acid in 0.5 M NaCl for 30 s (acid wash) prior to fixation. Tfn633 (Invitrogen Molecular Probes) internalizations were conducted in serum-free DMEM, and cells were serum starved for 1 h prior to internalizations. Cells were fixed for 10 min in 2% formaldehyde in phosphate-buffered saline (PBS), rinsed in PBS, and incubated with primary antibodies in PBS/10% fetal calf serum in the presence (total) or absence (surface) of 0.2% saponin as indicated. Alexa dye–conjugated secondary antibodies were used to detect primary antibody localizations. All images were obtained using a 510 LSM confocal microscope (Zeiss, Thornwood, NY) with a 63× 1.3–numerical aperture PlanApo objective. MetaMorph (Molecular Devices, Sunnyvale, CA) was used to quantify colocalization. Individual cells were outlined, and the overlapping fluorescence pixels were quantified using the colocalization function. Adobe Photoshop (San Jose, CA) was used for image processing. Flow cytometry samples were run on an LSR2 cytometer with the help of the National Heart, Lung, and Blood Institute Flow Cytometry Core Facility. Data analysis was performed using FloJo Software (Tree Star, Ashland, OR).
Whole-cell lysis and immunoprecipitation
Sixty-millimeter plates of transfected HeLa cells were washed twice in cold PBS and scraped into lysis buffer (1% IGEPAL [Sigma-Aldrich], 10% glycerol, 50 mM Tris, pH 7.5, 100 mM NaCl) with complete Mini Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and 20 μM N-ethylmaleimide (Sigma-Aldrich). Samples were sonicated three times for 5-s pulses at 4°C with a Microson Ultrasonic Cell Disruptor (Misonix, Newtown, CT), power level 2. Samples were centrifuged at 1300 × g for 10 min at 4°C and supernatants removed. For immunoprecipitations, protein G–Sepharose beads (GE Healthcare, Uppsala, Sweden) were prepared per manufacturer's instructions, and lysates were added with 5 μg of anti-CD98 antibody (clone MEM-108). Samples were rocked end over end for 1 h at 4°C. Samples were washed four times with Lysis buffer and solubilized in SDS–PAGE sample buffer. Samples were run on 4–20% Tris-HCl polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose (Whatman, Sanford, ME). Western blots were probed with designated antibodies and analyzed using n Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE) and accompanying software.
CD98-SNAP immunofluorescence and immunoprecipitation
CD98-SNAP was constructed by fusing the SNAP open reading frame (pSEMS1-26m from Covalys/New England Biolabs, Ipswich, MA) onto the C-terminus of the type II protein CD98 (4F2 cell-surface antigen heavy-chain isoform b) by standard two-stage PCR protocols. A (GGGS)2 linker was placed between CD98 and the SNAP tag. The PCR fusion was cloned into the TA mammalian expression vector pTarget (Clontech, Mountain View, CA).
For immunofluorescence, SNAP-Surface-488 (New England Biolabs) (called here BG-488) was added to media to label the surface of HeLa cells expressing CD98-SNAP (with and without MARCH8 cotransfection), and the cells were then incubated at 37°C for 1 h. Cells were then washed and transferred to fresh media containing 15 mM NH4Cl and treated as described earlier.
For pull-down of CD98-SNAP, a non–cell-permeable BG substrate was prepared by reacting O6-[4-(aminomethyl)benzyl]guanine (BG-NH2) (1.3 Eq; Toronto Research Chemicals, North York, Canada) with EZ-Link NHS-PEG4-Biotin (Thermo Scientific, Waltham, MA). Reactions were performed at 30°C for 16 h in N,N-dimethylacetamide in the presence of 2 Eq triethylamine. BG-PEG4-Biotin was analyzed by high-resolution mass spectrometry and purity checked by high-performance liquid chromatography.
For expression, HeLa cells (10-cm dishes) were transfected with CD98-SNAP with or without HA-ubiquitin and MARCH8-FLAG. After 18 h, cells were labeled with BG-PEG4-Biotin substrate (3 mM) for up to 4 h at 37°C. Cells were rinsed three times with PBS, lifted, and pelleted at 300 × g. Cell pellets were incubated with 200 mM BG-NH2 for 15 min at 4°C, then solubilized in 0.2 ml of lysis buffer (50 mM Tris-Cl, pH 7.4, 0.25 M NaCl, 0.1% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM dithiothreitol, and 100 mM Na3VO4) plus Complete Protease Inhibitor tablets (Roche, Indianapolis, IN). The cell extract was denatured by adding SDS to obtain a 1% final concentration and boiled for 15 min. The SDS was quenched by adding 0.1 ml of 20% Triton X-100 and 1.8 ml of lysis buffer and placed on ice for 30 min. The lysate was centrifuged at 13,000 × g for 5 min, and 50 μl of a 1:1 slurry of streptavidin–agarose (Sigma-Aldrich) was added to the supernatant. The lysate was rocked at room temperature for 1 h, and the beads were washed three times with lysis buffer and once with water. Thirty microliters of 2× SDS sample buffer was added, and the beads were boiled for 10 min before protein separation by SDS–PAGE (4–12% Tris-glycine; Novex, Invitrogen, Eugene, OR), transfer to nitrocellulose, and immunoblotting.
HA-ubiquitin was detected with monoclonal HA.11 (Covance). Alexa Fluor 680 secondary antibodies (Invitrogen) were used for HA detection. Biotinylated CD98-SNAP was detected with NeutrAvidin, DyLight-800 Conjugated (Thermo Scientific). The membrane was incubated with primary and secondary antibodies, each for 1 h at room temperature. The membrane was washed three times with 0.1% Tween 20 in phosphate-buffered saline and then quantitatively visualized by scanning with an Odyssey infrared scanner (Li-Cor Biosciences,).